?Ceramide-1-phosphate (C1P) protects against cyclophosphamide-induced gonadotoxicity in a mouse model

OUBIÑA G,; N PASCUALI; FERNANDA PARBORELL; DI PIETRO, M.; ABRAMOVICH D; L SCOTTI

Rome

Congreso; 17 th World Congress of the Academy of Human Reproduction; 2017

17 th World Congress of the Academy of Human Reproduction

We investigated whether the administration of the sphingolipid ceramide-1-phosphate (C1P) can preserve ovarian function from gonadotoxicity caused by cyclophosphamide (CTX) in a mouse model. C1P protects against CTX-induced gonadotoxicity by preserving the ovarian reserve and contributing to blood vessel formation, providing a novel strategy to avoid chemotherapy-induced POF.Premature ovarian failure (POF) is often a consequence of gonadotoxic chemoradiotherapy. Depleted follicle reserve can present with transient or permanent amenorrhea, infertility and premature menopause. In particular, alkylating agents such as cyclophosphamide (CTX) induce severe follicle loss, the proposed mechanisms being apoptosis and/or damage to ovarian microvascularization. Bioactive sphingolipids, such as C1P, are important regulators of cell homeostasis. As well as sphingosine-1-phosphate (S1P), C1P has a proangiogenic and anti-apoptotic role, with the advantage of a greater stability. Since the efficacy of gonadotropin-releasing hormone analogs is still controversial, we propose C1P as a potential protective molecule for fertility preservation. Based on these considerations, we investigated whether the administration of C1P can preserve ovarian function from gonadotoxicity caused by cyclophosphamide (CTX) in a mouse model. Twenty-four 8-week-old female F1 mice (BALB/c x C57BL/6) were separated into four groups (n=6/group). Mice received either a single intraperitoneal injection of saline (control group) or 75 mg/kg of CTX. Besides, CTX mice received intrabursal administration of vehicle (CTX group) or C1P in two different doses (CTX+C1P groups: 5ul/ovary; 0.5 mM or 10ul/ovary; 0.6 mM). Animals were euthanized on day 14, and their ovaries removed and fixed in Bouin?s solution for further study. Bouin-fixed, paraffin-embedded slides of ovarian tissue were stained with hematoxylin and eosin (H&E) and the number of different stages of follicles was determined in 3 sections per ovary. Data are expressed as the percentage of each follicle type per ovary. Ovaries were also immunostained for anti-müllerian hormone (AMH) and for vonWillebrand factor by immunohistochemistry (IHC). Quantification of relative vascular areas was performed with ImageProPlus software. Firstly, we analyzed follicular structures in H&E-stained ovarian slides. In CTX-treated ovaries, percentages of primary and preantral follicles were lower (both p